The invention relates to methods for the detection, diagnosis, prognosis and treatment of cancer. Specifically, the invention describes the detection of microtubule associated proteinxe2x80x942 (MAP-2) in tumor cells, and the use of MAP-2 as an indicator of metastatic potential. The invention also describes the use of MAP-2 to prevent non-metastatic primary tumors from progressing to later stage disease.
Publications referred to throughout the text of this document are incorporated by reference in their entireties in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Cancer is the second leading cause of death in the United States after heart disease (Boring et al., CA Cancer J. Clin., 43:7 (1993)). Cancer is characterized by the growth of abnormal (neoplastic) cells which develop from normal tissue. In cancer, cells acquire the ability to override normal constraints on the cell and proliferate under conditions in which normal cells would not grow. Among the most potent cancer causing agents (carcinogens) are ultraviolet and ionizing forms of radiation. Thus, cancer is a typically a disease of malfunctioning cellular genes (or unwanted viral expression) which leads to unchecked growth of tissue. It is generally accepted that multiple mutations must occur to cause cancer, and that cells must progress through several steps in the transformation from normal cells to an early-stage tumor and finally, to invasive and metastatic disease.
Cancer arises from a deregulation in the communication between tumor cells and their environment. Normally, cells do not divide in the absence of stimulatory signals. In cancer, this control is lost, and the cell is able to override its normal quiescent state and proliferate without restriction. Once a tumor reaches a certain size, however, growth is limited by diffusion of nutrients through the tumor. At this point tumors may generate angiogenic factors which promote neovascularization of the tumor and allow for tumor cells to be carried to distant sites in a process known as metastasis. A variety of factors have been implicated in metastasis including growth factors, cell surface receptors, and cytokines which modulate cell division.
For example, melanoma is a type of skin cancer which, if not removed while confined to the upper layers of the skin, spreads internally and is usually fatal. There are four stages of melanoma. Stages I and II are early stages of the disease, and are classified according to the thickness of the tumor, known as the Breslow""s thickness, and by the number of layers of skin invaded by the tumor, known as the Clark""s level of invasion (see e.g. Robbins, P. and Perez, M., Understanding Melanoma: What You Need to Know, The Skin Cancer Foundation (1996)). Stages III and IV, the more advanced stages of the disease, are classified according to the degree of melanoma spread beyond the skin. By Stage III, the tumor has metastasized to lymph nodes in the region of the original tumor or nearby skin. At this point, Breslow thickness and Clark""s level are no longer described in the diagnosis. Finally, by Stage IV, the melanoma has metastasized to lymph nodes far away from the primary tumor or to internal organs, such as the lung, liver, brain, bone and gastrointestinal tract. Later stages of cancer generally require that alternative diagnostic and preventative tools be invoked. For example, Stage III melanoma may require evaluation of the lymph nodes. With distant metastases (i.e. Stage IV), the physician may call for imaging of the head, chest, abdomen and pelvis. Finally, chemotherapy and immunotherapy treatments may be invoked.
Methods for the general detection of genes for cancer diagnosis and prevention of tumor progression are described in U.S. Pat. Nos. 6,025,137, 5,674,739 and 5,633,161, which describe methods for detection of genes differentially expressed in cancer and the identification of mouse and human fomy030 gene in melanoma cells. For example, with respect to melanoma, markers include tyrosinase (U.S. Pat. No. 6,153,388), and tyrosinase, MART-1, and MAGE-3 (U.S. Pat. No. 6,057,105). Additionally, the use of gp75 antigen (TRP-1) as a tumor vaccine for melanoma is described in U.S. Pat. No. 6,168,946, and the use of TRP-2 protein as a tumor antigen is described in U.S. Pat. No. 6,083,703.
In light of the increasing importance of cancer, there is a need to identify methods which will improve cancer detection and diagnostics. Also, there is a need for markers which may be used by a physician to establish a prognosis which includes an evaluation of the risk of metastatic disease. Such diagnostic markers may be highly significant in cancers such as melanoma, where tumors may be detected early. Also, there is a need to develop compounds which can prevent or reduce the transformation of primary (benign) tumors into metastatic disease.
The invention describes detection of microtubule associated proteinxe2x80x942 (MAP-2) protein in tumor cells and the use of MAP-2 as a marker to assess the metastatic potential of tumor cells. Specifically, the invention describes that a decrease in MAP-2 expression in tumors is associated with metastatic disease. In one aspect, the invention comprises a method for detecting MAP-2 protein in a sample comprising tumor cells comprising the steps of: obtaining a test sample comprising tumor cells; incubating the cells with a detection agent that forms a complex with MAP-2 protein or fragments thereof; and detecting the presence of MAP-2 protein. The cellular membrane of the cells may be disrupted to allow access to intracellular proteins. In embodiments of the present invention, the presence of MAP-2 protein may be detected by detecting the presence of a complex formed between the detection agent and the MAP-2 protein.
It may be preferable to measure MAP-2 expression as levels of MAP-2 mRNA or cDNA in tumor cells. Thus, in another aspect, the invention comprises a method for detecting MAP-2 mRNA in a sample comprising tumor cells comprising the steps of: obtaining a sample comprising tumor cells; exposing the sample under high stringency conditions to at least one isolated nucleic acid that hybridizes to MAP-2 mRNA; and determining the levels MAP-2 mRNA based on the hybridization of the nucleic acid to MAP-2 mRNA.
In another aspect, the invention comprises a method for determining the metastatic potential of a tumor sample comprising the steps of: obtaining a test tumor sample of unknown metastatic potential from a subject; determining the level of MAP-2 expression in the test sample; and assessing the metastatic potential of the sample based on the level of MAP-2 expression in the test sample relative to a non-metastatic control comprising cells of the same or a similar tumor type.
Because an increase MAP-2 expression is associated with reduced metastatic potential, the invention contemplates that modulators of MAP-2 expression comprise potential anti-tumor agents. Thus, in another aspect, the invention comprises a method for screening for compounds that modulate MAP-2 expression comprising: incubating cells with a compound of interest; disrupting the cellular membrane of the cells to allow access to intracellular proteins; incubating the cell proteins with a detection agent which recognizes and complexes with MAP-2 protein or fragments thereof; detecting the presence of the complex; and comparing the levels of MAP-2 protein in cells treated with the compound of interest to the levels of MAP-2 protein in cells not incubated with the compound of interest.
Alternatively, the method of screening may measure MAP-2 mRNA or cDNA levels as a means of screening for compounds that modulate MAP-2 expression. Thus, in another aspect, the invention comprises a method for screening for compounds that modulate MAP-2 expression comprising: incubating cells with a compound of interest; exposing the cells under high stringency conditions to at least one isolated nucleic acid that hybridizes to MAP-2 mRNA; and determining the levels of MAP-2 mRNA based on the hybridization of the nucleic acid to MAP-2 mRNA; and comparing the levels of MAP-2 mRNA in cells treated with the compound of interest as compared to cells not incubated with the compound of interest.
In that expression of MAP-2 is strongest in primary tumors, with a decrease as cells progress to later stages of cancer, MAP-2 may be used to prevent early stage tumors from progressing to later stage diseases. Thus, in another aspect, the invention comprises a method to prevent tumor progression in metastatic melanoma comprising increasing levels of MAP-2 expression in cells.
In yet another aspect, the invention includes the development of kits comprising reagents for performing the methods of the invention.
The foregoing focuses on the more important features of the invention in order that the detailed description which follows may be better understood and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention which will be described hereinafter and which will form the subject matter of the claims appended hereto. It is to be understood that the invention is not limited in its application to the components set forth in the following description and drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways.
From the foregoing summary, it is apparent that an object of the present invention is to provide a method for the use of MAP-2 as a marker to assess the metastatic potential of tumors such as melanoma and other tumors of neural crest derived tissue. The invention also describes the use of MAP-2 to prevent tumor progression by increasing levels of MAP-2 protein in cells. These, together with other objects of the present invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this document.